Inhibitors of histone deacetylase

ABSTRACT

This invention comprises the novel compounds of formula (I) 
     
       
         
         
             
             
         
       
     
     wherein n, R 1 , R 2 , R 3 , R 4 , Q, X, Y, Z and 
     
       
         
         
             
             
         
       
     
     have defined meanings, having histone deacetylase inhibiting enzymatic activity; their preparation, compositions containing them and their use as a medicine.

This invention concerns compounds having histone deacetylase (HDAC)inhibiting enzymatic activity. It further relates to processes for theirpreparation, to compositions comprising them, as well as their use, bothin vitro and in vivo, to inhibit HDAC and as a medicine, for instance asa medicine to inhibit proliferative conditions, such as cancer andpsoriasis.

In all eukaryotic cells, genomic DNA in chromatine associates withhistones to form nucleosomes. Each nucleosome consists of a proteinoctamer made up of two copies of each histones H2A, H2B, H3 and H4. DNAwinds around this protein core, with the basic amino acids of thehistones interacting with the negatively charged phosphate groups of theDNA. The most common posttranslational modification of these corehistones is the reversible acetylation of the ε-amino groups ofconserved, highly basic N-terminal lysine residues. The steady state ofhistone acetylation is established by the dynamic equilibrium betweencompeting histone acetyltransferase(s) and histone deacetylase(s) hereinreferred to as “HDAC”. Histone acetylation and deacetylation has longbeen linked to transcriptional control. The recent cloning of the genesencoding different histone acetyltransferases and histone deacetylasesprovided a possible explanation for the relationship between histoneacetylation and transcriptional control. The reversible acetylation ofhistones can result in chromatin remodelling and as such act as acontrol mechanism for gene transcription. In general, hyperacetylationof histones facilitates gene expression, whereas histone deacetylationis correlated with transcriptional repression. Histoneacetyltransferases were shown to act as transcriptional coactivators,whereas histone deacetylases were found to belong to transcriptionalrepression pathways.

The dynamic equilibrium between histone acetylation and deacetylation isessential for normal cell growth. Inhibition of histone deacetylaseresults in cell cycle arrest, cellular differentiation, apoptosis andreversal of the transformed phenotype. Therefore HDAC inhibitors canhave great therapeutic potential in the treatment of cell proliferativediseases or conditions (Marks et al., Nature Reviews: Cancer 1: 194-202,2001)

The study of inhibitors of histone deacetylases (HDAC) indicates thatindeed these enzymes play an important role in cell proliferation anddifferentiation. The inhibitor Trichostatin A (TSA) causes cell cyclearrest at both G1 and G2 phases, reverts the transformed phenotype ofdifferent cell lines, and induces differentiation of Friend leukemiacells and others. TSA (and suberoylanilide hydroxamic acid SAHA) havebeen reported to inhibit cell growth, induce terminal differentiation,and prevent the formation of tumours in mice (Finnin et al., Nature,401: 188-193, 1999).

Trichostatin A has also been reported to be useful in the treatment offibrosis, e.g. liver fibrosis and liver chirrhosis. (Geerts et al.,European Patent Application EP 0 827 742, published 11 Mar. 1998).

Patent application WO01/38322 published on May 31, 2001 disclosesamongst others inhibitors of histone deacetylase of general formulaCy-L¹-Ar—Y¹—C(O)—NH-Z, providing compositions and methods for treatingcell proliferative diseases and conditions.

Patent application WO01/70675 published on 27 Sep. 2001 disclosesinhibitors of histone deacetylase of formula Cy-X—Y¹—W andCy-S(O)₂—NH—Y³—W and further provides compositions and methods fortreating cell proliferative diseases and conditions.

The problem to be solved is to provide histone deacetylase inhibitorswith high enzymatic activity and also show advantageous properties suchas cellular activity and increased bioavailability, preferably oralbioavailability, and have little or no side effects.

The novel compounds of the present invention solve the above describedproblem. The compounds differ from the prior art in structure.

The compounds of the present invention show excellent in-vitro histonedeacetylase inhibiting enzymatic activity. The present compounds haveadvantageous properties with regard to cellular activity and specificproperties with regard to inhibition of cell cycle progression at bothG1 and G2 checkpoints (p21 induction capacity). The compounds of thepresent invention show good metabolic stability and high bioavailabilityand more particular they show oral bioavailability.

This invention concerns compounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

n is 0, 1, 2 or 3 and when n is 0 then a direct bond is intended;

each Q is nitrogen or

each X is nitrogen or

each Y is nitrogen or

each Z is nitrogen or

-   R¹ is —C(O)NR⁵R⁶, —N(H)C(O)R⁷, —C(O)—C₁₋₆alkanediylSR⁷,    —NR⁸C(O)N(OH)R⁷, —NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R⁷ or    another Zn-chelating-group wherein R⁵ and R⁶ are each independently    selected from hydrogen, hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkyl,    aminoC₁₋₆alkyl or aminoaryl;    -   R⁷ is independently selected from hydrogen, C₁₋₆alkyl,        C₁₋₆alkylcarbonyl, arylC₁₋₆alkyl, C₁₋₆alkylpyrazinyl,        pyridinone, pyrrolidinone or methylimidazolyl;    -   R⁸ is independently selected from hydrogen or C₁₋₆alkyl;-   R² is hydrogen, halo, hydroxy, amino, nitro, C₁₋₆alkyl,    C₁₋₆alkyloxy, trifluoromethyl, di(C₁₋₆alkyl)amino, hydroxyamino or    naphtalenylsulfonylpyrazinyl;-   R³ is hydrogen, C₁₋₆alkyl, arylC₂₋₆alkenediyl, furanylcarbonyl,    naphtalenylcarbonyl, —C(O)phenylR⁹, C₁₋₆alkylaminocarbonyl,    aminosulfonyl, arylaminosulfonyl, aminosulfonylamino,    di(C₁₋₆alkyl)aminosulfonylamino, arylaminosulfonylamino,    aminosulfonylaminoC₁₋₆alkyl,    di(C₁₋₆alkyl)aminosulfonylaminoC₁₋₆alkyl,    arylaminosulfonylaminoC₁₋₆alkyl, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,    C₁₋₁₂alkylsulfonyl, di(C₁₋₆alkyl)aminosulfonyl,    trihaloC₁₋₆alkylsulfonyl, di(aryl)C₁₋₆alkylcarbonyl,    thiophenylC₁₋₆alkylcarbonyl, pyridinylcarbonyl or    arylC₁₋₆alkylcarbonyl    -   wherein each R⁹ is independently selected from phenyl; phenyl        substituted with one, two or three substituents independently        selected from halo, amino, C₁₋₆alkyl, C₁₋₆alkyloxy,        hydroxyC₁₋₄alkyl, hydroxyC₁₋₄alkyloxy, aminoC₁₋₄alkyloxy,        di(C₁₋₄alkyl)aminoC₁₋₄alkyloxy, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,        di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl,        hydroxyC₁₋₄alkylpiperazinylC₁₋₄alkyl,        C₁₋₄alkyloxypiperidinylC₁₋₄alkyl,        hydroxyC₁₋₄alkyloxyC₁₋₄alkylpiperazinyl,        C₁₋₄alkylpiperazinylC₁₋₄alkyl,        di(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkyl, pyrrolidinylC₁₋₄alkyloxy,        morpholinylC₁₋₄alkyloxy, or morpholinylC₁₋₄alkyl; thiophenyl; or        thiophenyl substituted with di(C₁₋₄alkyl)aminoC₁₋₄alkyloxy,        di(C₁₋₆alkyl)aminoC₁₋₆alkyl,        di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl,        pyrrolidinylC₁₋₄alkyloxy, C₁₋₄alkylpiperazinylC₁₋₄alkyl,        di(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkyl or morpholinylC₁₋₄alkyloxy.-   R⁴ is hydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl,    C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl, hydroxycarbonyl,    aminoC₁₋₆alkyl, aminocarbonylC₁₋₆alkyl, hydroxycarbonylC₁₋₆alkyl,    hydroxyaminocarbonyl, C₁₋₆alkyloxycarbonyl, C₁₋₆alkylaminoC₁₋₆alkyl    or di(C₁₋₆alkyl)aminoC₁₋₆alkyl;-   when R³and R⁴ are present on the same carbon atom, R³and R⁴ together    may form a bivalent radical of formula

—C(O)—NH—CH₂—NR¹⁰—  (a-1)

-   -   wherein R¹⁰ is hydrogen or aryl;

-   when R³ and R⁴ are present on adjacent carbon atoms, R³ and R⁴    together may form a bivalent radical of formula

═CH—CH═CH—CH═  (b-1);

-   aryl in the above is phenyl, or phenyl substituted with one or more    substituents each independently selected from halo, C₁₋₆alkyl,    C₁₋₆alkyloxy, trifluoromethyl, cyano or hydroxycarbonyl.

The term “histone deacetylase inhibitor” or “inhibitor of histonedeacetylase” is used to identify a compound, which is capable ofinteracting with a histone deacetylase and inhibiting its activity, moreparticularly its enzymatic activity. Inhibiting histone deacetylaseenzymatic activity means reducing the ability of a histone deacetylaseto remove an acetyl group from a histone. Preferably, such inhibition isspecific, i.e. the histone deacetylase inhibitor reduces the ability ofa histone deacetylase to remove an acetyl group from a histone at aconcentration that is lower than the concentration of the inhibitor thatis required to produce some other, unrelated biological effect.

As used in the foregoing definitions and hereinafter, halo is generic tofluoro, chloro, bromo and iodo; C₁₋₄alkyl defines straight and branchedchain saturated hydrocarbon radicals having from 1 to 4 carbon atomssuch as, e.g. methyl, ethyl, propyl, butyl, 1-methylethyl,2-methylpropyl and the like; C₁₋₆alkyl includes C₁₋₄alkyl and the higherhomologues thereof having 5 to 6 carbon atoms such as, for example,pentyl, 2-methyl-butyl, hexyl, 2-methylpentyl and the like;C₁₋₆alkanediyl defines bivalent straight and branched chained saturatedhydrocarbon radicals having from 1 to 6 carbon atoms such as, forexample, methylene, 1,2-ethanediyl, 1,3-propanediyl 1,4-butanediyl,1,5-pentanediyl, 1,6-hexanediyl and the branched isomers thereof suchas, 2-methylpentanediyl, 3-methylpentanediyl, 2,2-dimethylbutanediyl,2,3-dimethylbutanediyl and the like; trihaloC₁₋₆alkyl defines C₁₋₆alkylcontaining three identical or different halo substituents for exampletrifluoromethyl; C₂₋₆alkenediyl defines bivalent straight and branchedchain hydrocarbon radicals containing one double bond and having from 2to 6 carbon atoms such as, for example, ethenediyl, 2-propenediyl,3-butenediyl, 2-pentenediyl, 3-pentenediyl, 3-methyl-2-butenediyl, andthe like; and aminoaryl defines aryl substituted with amino.

The term “another Zn-chelating group” refers to a group, which iscapable of interacting with a Zn-ion, which can be present at anenzymatic binding site.

Pharmaceutically acceptable addition salts encompass pharmaceuticallyacceptable acid addition salts and pharmaceutically acceptable baseaddition salts. The pharmaceutically acceptable acid addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid addition salt forms, which the compounds of formula (I)are able to form. The compounds of formula (I) which have basicproperties can be converted in their pharmaceutically acceptable acidaddition salts by treating said base form with an appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric;nitric; phosphoric and the like acids; or organic acids such as, forexample, acetic, trifluoroacetic, propanoic, hydroxyacetic, lactic,pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-amino-salicylic, pamoic and the like acids.

The compounds of formula (I) which have acidic properties may beconverted in their pharmaceutically acceptable base addition salts bytreating said acid form with a suitable organic or inorganic base.Appropriate base salt forms comprise, for example, the ammonium salts,the alkali and earth alkaline metal salts, e.g. the lithium, sodium,potassium, magnesium, calcium salts and the like, salts with organicbases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, andsalts with amino acids such as, for example, arginine, lysine and thelike.

The term “acid or base addition salts” also comprises the hydrates andthe solvent addition forms, which the compounds of formula (I) are ableto form. Examples of such forms are e.g. hydrates, alcoholates and thelike.

The term “stereochemically isomeric forms of compounds of formula (I)”,as used herein, defines all possible compounds made up of the same atomsbonded by the same sequence of bonds but having differentthree-dimensional structures, which are not interchangeable, which thecompounds of formula (I) may possess. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms, which saidcompound might possess. Said mixture may contain all diastereomersand/or enantiomers of the basic molecular structure of said compound.All stereochemically isomeric forms of the compounds of formula (I) bothin pure form or in admixture with each other are intended to be embracedwithin the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprisethose compounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide, particularly those N-oxides whereinone or more of the piperidine-, piperazine or pyridazinyl-nitrogens areN-oxidized.

Some of the compounds of formula (I) may also exist in their tautomericforms. Such forms although not explicitly indicated in the above formulaare intended to be included within the scope of the present invention.

Whenever used hereinafter, the term “compounds of formula (I)” is meantto include also the pharmaceutically acceptable addition salts and allstereoisomeric forms.

As used herein, the terms “histone deacetylase” and “HDAC” are intendedto refer to any one of a family of enzymes that remove acetyl groupsfrom the ε-amino groups of lysine residues at the N-terminus of ahistone. Unless otherwise indicated by context, the term “histone” ismeant to refer to any histone protein, including H1, H2A, H2B, H3, H4,and H5, from any species. Human HDAC proteins or gene products, include,but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6,HDAC-7, HDAC-8, HDAC-9 and HDAC-10. The histone deacetylase can also bederived from a protozoal or fungal source.

A first group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) n is 0 or 1;-   b) each Q is

-   c) R¹ is —C(O)NH(OH) or —NHC(O)C₁₋₆alkanediylSH;-   d) R² is hydrogen or nitro;-   e) R³ is C₁₋₆alkyl, arylC₂₋₆alkenediyl, furanylcarbonyl,    naphtalenylcarbonyl, C₁₋₆alkylaminocarbonyl, aminosulfonyl,    di(C₁₋₆alkyl)aminosulfonylaminoC₁₋₆alkyl,    di(C₁₋₆alkyl)aminoC₁₋₆alkyl, C₁₋₁₂alkylsulfonyl,    di(C₁₋₆alkyl)aminosulfonyl, trihaloC₁₋₆alkylsulfonyl,    di(aryl)C₁₋₆alkylcarbonyl, thiophenylC₁₋₆alkylcarbonyl,    pyridinylcarbonyl or arylC₁₋₆alkylcarbonyl;-   f) R⁴ is hydrogen;-   g) when R³ and R⁴ are present on the same carbon atom R³ and R⁴    together may form a bivalent radical of formula (a-1) wherein R¹⁰ is    aryl;-   h) when R³ and R⁴ are present on adjacent carbon atoms R³ and R⁴    together may form a bivalent radical of formula (b-1).

A second group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) n is 1;-   b) each Q is

-   c) each Z is nitrogen;-   d) R¹ is —C(O)NH(OH);-   e) R² is hydrogen;-   f) R³ is naphtalenylcarbonyl, C₁₋₁₂alkylsulfonyl or    di(aryl)C₁₋₆alkylcarbonyl;-   g) R⁴ is hydrogen.

A third group of interesting compounds consists of those compounds offormula (I) wherein R² is hydrogen.

A fourth group of interesting compounds consists of those compounds offormula (I) wherein R¹ is —C(O)NH(OH).

A fifth group of interesting compounds consists of those compounds offormula (I) wherein R² is hydrogen and R¹ is —C(O)NH(OH).

A sixth group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply;

-   a) R¹ is —C(O)NR⁵R⁶, —C(O)—C₁₋₆alkanediylSR⁷, —NR⁸C(O)N(OH)R⁷,    —NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R⁷ or another    Zn-chelating-group wherein R⁵ and R⁶ are each independently selected    from hydrogen, hydroxy, hydroxyC₁₋₆alkyl or aminoC₁₋₆alkyl;-   b) R² is hydrogen, halo, hydroxy, amino, nitro, C₁₋₆alkyl,    C₁₋₆alkyloxy, trifluoromethyl or di(C₁₋₆alkyl)amino;-   c) R³ is hydrogen, C₁₋₆alkyl, arylC₂₋₆alkenediyl, furanylcarbonyl,    naphtalenylcarbonyl, —C(O)phenylR⁹, C₁₋₆alkylaminocarbonyl,    aminosulfonyl, arylaminosulfonyl, aminosulfonylamino,    di(C₁₋₆alkyl)aminosulfonylamino, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,    C₁₋₁₂alkylsulfonyl, di(C₁₋₆alkyl)aminosulfonyl or pyridinylcarbonyl    wherein each R⁹ is independently selected from phenyl; phenyl    substituted with one, two or three substituents independently    selected from halo, C₁₋₆alkyl, C₁₋₆alkyloxy; or thiophenyl;-   d) R⁴ is hydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl,    C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl, aminoC₁₋₆alkyl,    C₁₋₆alkylaminoC₁₋₆alkyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl.

A group of preferred compounds consists of those compounds of formula(I) wherein

-   R¹ is —C(O)NR⁵R⁶, —C(O)—C₁₋₆alkanediylSR⁷, —NR⁸C(O)N(OH)R⁷,    —NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R⁷ or another    Zn-chelating-group wherein R⁵ and R⁶ are each independently selected    from hydrogen, hydroxy, hydroxyC₁₋₆alkyl or aminoC₁₋₆alkyl;-   R² is hydrogen, halo, hydroxy, amino, nitro, C₁₋₆alkyl,    C₁₋₆alkyloxy, trifluoromethyl or di(C₁₋₆alkyl)amino;-   R³ is hydrogen, C₁₋₆alkyl, arylC₂₋₆alkenediyl, furanylcarbonyl,    naphtalenylcarbonyl, —C(O)phenylR⁹, C₁₋₆alkylaminocarbonyl,    aminosulfonyl, arylaminosulfonyl, aminosulfonylamino,    di(C₁₋₆alkyl)aminosulfonylamino, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,    C₁₋₁₂alkylsulfonyl, di(C₁₋₆alkyl)aminosulfonyl or pyridinylcarbonyl    wherein each R⁹ is independently selected from phenyl; phenyl    substituted with one, two or three substituents independently    selected from halo, C₁₋₆alkyl, C₁₋₆alkyloxy; or thiophenyl; and-   R⁴ is hydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl,    C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl, aminoC₁₋₆alkyl,    C₁₋₆alkylaminoC₁₋₆alkyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl.

A further group of preferred compounds consists of those compounds offormula (I)

-   -   wherein n is 0 or 1; each Q is

-   -   R¹ is —C(O)NH(OH) or —NHC(O)C₁₋₆alkanediylSH; R² is hydrogen or        nitro; R³is C₁₋₆alkyl, arylC₂₋₆alkenediyl, furanylcarbonyl,        naphtalenylcarbonyl, C₁₋₆alkylaminocarbonyl, aminosulfonyl,        di(C₁₋₆alkyl)aminosulfonylaminoC₁₋₆alkyl,        di(C₁₋₆alkyl)aminoC₁₋₆alkyl, C₁₋₁₂alkylsulfonyl,        di(C₁₋₆alkyl)aminosulfonyl, trihaloC₁₋₆alkylsulfonyl,        di(aryl)C₁₋₆alkylcarbonyl, thiophenylC₁₋₆alkylcarbonyl,        pyridinylcarbonyl or arylC₁₋₆alkylcarbonyl; R⁴ is hydrogen; when        R³ and R⁴ are present on the same carbon atom R³ and R⁴ together        may form a bivalent radical of formula (a-1) wherein R¹⁰ is        aryl; or when R³ and R⁴ are present on adjacent carbon atoms R³        and R⁴ together may form a bivalent radical of formula (b-1).

A group of more preferred compounds consists of those compounds offormula (I)

-   -   wherein n is 1; each Q is

each Z is nitrogen; R¹ is —C(O)NH(OH); R² is hydrogen; R³isnaphtalenylcarbonyl, C₁₋₁₂alkylsulfonyl or di(aryl)C₁₋₆alkylcarbonyl;and R⁴is hydrogen.

Most preferred compounds are compounds No. 18, No. 5 and No. 24.

The compounds of formula (I) and their pharmaceutically acceptable saltsand N-oxides and stereochemically isomeric forms thereof may be preparedin conventional manner.

A general synthesis route is encompassed as example:

a) Hydroxamic acids of formula (I) wherein R¹ is —C(O)NH(OH), saidcompounds being referred to as compounds of formula (I-a), may beprepared by reacting an intermediate of formula (II) with an appropriateacid, such as for example, trifluoro acetic acid. Said reaction isperformed in an appropriate solvent, such as, for example, methanol.

b) intermediates of formula (II) may be prepared by reacting anintermediate of formula (III) with an intermediate of formula (IV) inthe presence of appropriate reagents such asN′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). Thereaction may be performed in a suitable solvent such as a mixture of DCMand THF.

c) intermediates of formula (III) may be prepared by reacting anintermediate of formula (V) with an appropriate base such as NaOH in thepresence of a suitable solvent such as ethanol.

The compounds of formula (I) can also conveniently be prepared usingsolid phase synthesis techniques. In general, solid phase synthesisinvolves reacting an intermediate in a synthesis with a polymer support.This polymer-supported intermediate can then be carried on through anumber of synthesis steps. After each step, filtering the resin andwashing it numerous times with various solvents remove impurities. Ateach step the resin can be split up to react with various intermediatesin the next step thus allowing for the synthesis of a large number ofcompounds. After the last step in the procedure the resin is treatedwith a reagent or process to cleave the resin from the sample. Moredetailed explanation of the techniques used in solid phase chemistry isdescribed in for example “The Combinatorial Index” (B. Bunin, AcademicPress) and Novabiochem's 1999 Catalogue & Peptide Synthesis Handbook(Novabiochem AG, Switzerland) both incorporated herein by reference.

The compounds of formula (I) and some of the intermediates may have atleast one stereogenic centre in their structure. This stereogenic centremay be present in an R or an S configuration.

The compounds of formula (I) as prepared in the hereinabove describedprocesses are generally racemic mixtures of enantiomers, which can beseparated from one another following art-known resolution procedures.The racemic compounds of formula (I) may be converted into thecorresponding diastereomeric salt forms by reaction with a suitablechiral acid. Said diastereomeric salt forms are subsequently separated,for example, by selective or fractional crystallization and theenantiomers are liberated there from by alkali. An alternative manner ofseparating the enantiomeric forms of the compounds of formula (I)involves liquid chromatography using a chiral stationary phase. Saidpure stereochemically isomeric forms may also be derived from thecorresponding pure stereochemically isomeric forms of the appropriatestarting materials, provided that the reaction occursstereospecifically. Preferably if a specific stereoisomer is desired,said compound would be synthesized by stereospecific methods ofpreparation. These methods will advantageously employ enantiomericallypure starting materials.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof have valuablepharmacological properties in that they have a histone deacetylase(HDAC) inhibitory effect.

This invention provides a method for inhibiting the abnormal growth ofcells, including transformed cells, by administering an effective amountof a compound of the invention. Abnormal growth of cells refers to cellgrowth independent of normal regulatory mechanisms (e.g. loss of contactinhibition). This includes the inhibition of tumour growth both directlyby causing growth arrest, terminal differentiation and/or apoptosis ofcancer cells, and indirectly, by inhibiting neovascularization oftumours.

This invention also provides a method for inhibiting tumour growth byadministering an effective amount of a compound of the presentinvention, to a subject, e.g. a mammal (and more particularly a human)in need of such treatment. In particular, this invention provides amethod for inhibiting the growth of tumours by the administration of aneffective amount of the compounds of the present invention. Examples oftumours which may be inhibited, but are not limited to, lung cancer(e.g. adenocarcinoma and including non-small cell lung cancer),pancreatic cancers (e.g. pancreatic carcinoma such as, for exampleexocrine pancreatic carcinoma), colon cancers (e.g. colorectalcarcinomas, such as, for example, colon adenocarcinoma and colonadenoma), prostate cancer including the advanced disease, hematopoietictumours of lymphoid lineage (e.g. acute lymphocytic leukemia, B-celllymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acutemyelogenous leukemia (AML)), thyroid follicular cancer, myelodysplasticsyndrome (MDS), tumours of mesenchymal origin (e.g. fibrosarcomas andrhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas,gliomas, benign tumour of the skin (e.g. keratoacanthomas), breastcarcinoma (e.g. advanced breast cancer), kidney carcinoma, ovarycarcinoma, bladder carcinoma and epidermal carcinoma.

The compound according to the invention may be used for othertherapeutic purposes, for example:

-   -   a) the sensitisation of tumours to radiotherapy by administering        the compound according to the invention before, during or after        irradiation of the tumour for treating cancer;    -   b) treating arthropathies and osteopathological conditions such        as rheumatoid arthritis, osteoarthritis, juvenile arthritis,        gout, polyarthritis, psoriatic arthritis, ankylosing spondylitis        and systemic lupus erythematosus;    -   c) inhibiting smooth muscle cell proliferation including        vascular proliferative disorders, atherosclerosis and        restenosis;    -   d) treating inflammatory conditions and dermal conditions such        as ulcerative colitis, Crohn's disease, allergic rhinitis, graft        vs. host disease, conjunctivitis, asthma, ARDS, Behcets disease,        transplant rejection, uticaria, allergic dermatitis, alopecia        areata, scleroderma, exanthema, eczema, dermatomyositis, acne,        diabetes, systemic lupus erythematosis, Kawasaki's disease,        multiple sclerosis, emphysema, cystic fibrosis and chronic        bronchitis;    -   e) treating endometriosis, uterine fibroids, dysfunctional        uterine bleeding and endometrial hyperplasia;    -   f) treating ocular vascularisation including vasculopathy        affecting retinal and choroidal vessels;    -   g) treating a cardiac dysfunction;    -   h) inhibiting immunosuppressive conditions such as the treatment        of HIV infections;    -   i) treating renal dysfunction;    -   j) suppressing endocrine disorders;    -   k) inhibiting dysfunction of gluconeogenesis;    -   l) treating a neuropathology for example Parkinson's disease or        a neuropathology that results in a cognitive disorder, for        example, Alzheimer's disease or polyglutamine related neuronal        diseases;    -   m) inhibiting a neuromuscular pathology, for example,        amylotrophic lateral sclerosis;    -   n) treating spinal muscular atrophy;    -   o) treating other pathologic conditions amenable to treatment by        potentiating expression of a gene;    -   p) enhancing gene therapy.

Hence, the present invention discloses the compounds of formula (I) foruse as a medicine as well as the use of these compounds of formula (I)for the manufacture of a medicament for treating one or more of theabove mentioned conditions.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof can have valuablediagnostic properties in that they can be used for detecting oridentifying a HDAC in a biological sample comprising detecting ormeasuring the formation of a complex between a labelled compound and aHDAC.

The detecting or identifying methods can use compounds that are labelledwith labelling agents such as radioisotopes, enzymes, fluorescentsubstances, luminous substances, etc. Examples of the radioisotopesinclude ¹²⁵I, ¹³¹I, ³H and ¹⁴C. Enzymes are usually made detectable byconjugation of an appropriate substrate which, in turn catalyses adetectable reaction. Examples thereof include, for example,beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidaseand malate dehydrogenase, preferably horseradish peroxidase. Theluminous substances include, for example, luminol, luminol derivatives,luciferin, aequorin and luciferase.

Biological samples can be defined as body tissue or body fluids.Examples of body fluids are cerebrospinal fluid, blood, plasma, serum,urine, sputum, saliva and the like.

In view of their useful pharmacological properties, the subjectcompounds may be formulated into various pharmaceutical forms foradministration purposes.

To prepare the pharmaceutical compositions of this invention, aneffective amount of a particular compound, in base or acid addition saltform, as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, preferably, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets.

Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. For parenteralcompositions, the carrier will usually comprise sterile water, at leastin large part, though other ingredients, to aid solubility for example,may be included. Injectable solutions, for example, may be prepared inwhich the carrier comprises saline solution, glucose solution or amixture of saline and glucose solution. Injectable suspensions may alsobe prepared in which case appropriate liquid carriers, suspending agentsand the like may be employed. In the compositions suitable forpercutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not cause a significant deleterious effect to theskin. Said additives may facilitate the administration to the skinand/or may be helpful for preparing the desired compositions. Thesecompositions may be administered in various ways, e.g., as a transdermalpatch, as a spot-on or as an ointment.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used in thespecification and claims herein refers to physically discrete unitssuitable as unitary dosages, each unit containing a predeterminedquantity of active ingredient, calculated to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier. Examples of such dosage unit forms are tablets (includingscored or coated tablets), capsules, pills, powder packets, wafers,injectable solutions or suspensions, teaspoonfuls, tablespoonfuls andthe like, and segregated multiples thereof.

Those skilled in the art could easily determine the effective amountfrom the test results presented hereinafter. In general it iscontemplated that a therapeutically effective amount would be from 0.05mg/kg to 100 mg/kg body weight, and in particular from 0.05 mg/kg to 10mg/kg body weight. It may be appropriate to administer the required doseas two, three, four or more sub-doses at appropriate intervalsthroughout the day. Said sub-doses may be formulated as unit dosageforms, for example, containing 5 to 500 mg, and in particular 10 mg to500 mg of active ingredient per unit dosage form.

As another aspect of the present invention a combination of aHDAC-inhibitor with another anticancer agent is envisaged, especiallyfor use as a medicine, more specifically in the treatment of cancer orrelated diseases.

For the treatment of the above conditions, the compounds of theinvention may be advantageously employed in combination with one or moreother medicinal agents, more particularly, with other anti-canceragents. Examples of anti-cancer agents are:

-   -   platinum coordination compounds for example cisplatin,        carboplatin or oxalyplatin;    -   taxane compounds for example paclitaxel or docetaxel;    -   topoisomerase I inhibitors such as camptothecin compounds for        example irinotecan or topotecan;    -   topoisomerase II inhibitors such as anti-tumour podophyllotoxin        derivatives for example etoposide or teniposide;    -   anti-tumour vinca alkaloids for example vinblastine, vincristine        or vinorelbine;    -   anti-tumour nucleoside derivatives for example 5-fluorouracil,        gemcitabine or capecitabine;    -   alkylating agents such as nitrogen mustard or nitrosourea for        example cyclophosphamide, chlorambucil, carmustine or lomustine;    -   anti-tumour anthracycline derivatives for example daunorubicin,        doxorubicin, idarubicin or mitoxantrone;    -   HER2 antibodies for example trastuzumab;    -   estrogen receptor antagonists or selective estrogen receptor        modulators for example tamoxifen, toremifene, droloxifene,        faslodex or raloxifene;    -   aromatase inhibitors such as exemestane, anastrozole, letrazole        and vorozole;    -   differentiating agents such as retinoids, vitamin D and retinoic        acid metabolism blocking agents (RAMBA) for example accutane;    -   DNA methyl transferase inhibitors for example azacytidine;    -   kinase inhibitors for example flavoperidol, imatinib mesylate or        gefitinib;    -   farnesyltransferase inhibitors; or    -   other HDAC inhibitors.

The term “platinum coordination compound” is used herein to denote anytumor cell growth inhibiting platinum coordination compound whichprovides platinum in the form of an ion.

The term “taxane compounds” indicates a class of compounds having thetaxane ring system and related to or derived from extracts from certainspecies of yew (Taxus) trees.

The term “topisomerase inhibitors” is used to indicate enzymes that arecapable of altering DNA topology in eukaryotic cells. They are criticalfor important cellular functions and cell proliferation. There are twoclasses of topoisomerases in eukaryotic cells, namely type I and typeII. Topoisomerase I is a monomeric enzyme of approximately 100,000molecular weight. The enzyme binds to DNA and introduces a transientsingle-strand break, unwinds the double helix (or allows it to unwind)and subsequently reseals the break before dissociating from the DNAstrand. Topisomerase II has a similar mechanism of action which involvesthe induction of DNA strand breaks or the formation of free radicals.

The term “camptothecin compounds” is used to indicate compounds that arerelated to or derived from the parent camptothecin compound which is awater-insoluble alkaloid derived from the Chinese tree Camptothecinacuminata and the Indian tree Nothapodytes foetida.

The term “podophyllotoxin compounds” is used to indicate compounds thatare related to or derived from the parent podophyllotoxin, which isextracted from the mandrake plant.

The term “anti-tumor vinca alkaloids” is used to indicate compounds thatare related to or derived from extracts of the periwinkle plant (Vincarosea).

The term “alkylating agents” encompass a diverse group of chemicals thathave the common feature that they have the capacity to contribute, underphysiological conditions, alkyl groups to biologically vitalmacromolecules such as DNA. With most of the more important agents suchas the nitrogen mustards and the nitrosoureas, the active alkylatingmoieties are generated in vivo after complex degradative reactions, someof which are enzymatic. The most important pharmacological actions ofthe alkylating agents are those that disturb the fundamental mechanismsconcerned with cell proliferation in particular DNA synthesis and celldivision. The capacity of alkylating agents to interfere with DNAfunction and integrity in rapidly proliferating tissues provides thebasis for their therapeutic applications and for many of their toxicproperties.

The term “anti-tumour anthracycline derivatives” comprise antibioticsobtained from the fungus Strep. peuticus var. caesius and theirderivatives, characterised by having a tetracycline ring structure withan unusual sugar, daunosamine, attached by a glycosidic linkage.

Amplification of the human epidermal growth factor receptor 2 protein(HER 2) in primary breast carcinomas has been shown to correlate with apoor clinical prognosis for certain patients. Trastuzumab is a highlypurified recombinant DNA-derived humanized monoclonal IgG1 kappaantibody that binds with high affiniity and specificity to theextracellular domain of the HER2 receptor.

Many breast cancers have estrogen receptors and growth of these tumorscan be stimulated by estrogen. The terms “estrogen receptor antagonists”and “selective estrogen receptor modulators” are used to indicatecompetitive inhibitors of estradiol binding to the estrogen receptor(ER). Selective estrogen receptor modulators, when bound to the ER,induces a change in the three-dimensional shape of the receptor,inhibiting its binding to the estrogen responsive element (ERE) on DNA.

In postmenopausal women, the principal source of circulating estrogen isfrom conversion of adrenal and ovarian androgens (androstenedione andtestosterone) to estrogens (estrone and estradiol) by the aromataseenzyme in peripheral tissues. Estrogen deprivation through aromataseinhibition or inactivation is an effective and selective treatment forsome postmenopausal patients with hormone-dependent breast cancer.

The term “antiestrogen agent” is used herein to include not onlyestrogen receptor antagonists and selective estrogen receptor modulatorsbut also aromatase inhibitors as discussed above.

The term “differentiating agents” encompass compounds that can, invarious ways, inhibit cell proliferation and induce differentiation.Vitamin D and retinoids are known to play a major role in regulatinggrowth and differentiation of a wide variety of normal and malignantcell types. Retinoic acid metabolism blocking agents (RAMBA's) increasethe levels of endogenous retinoic acids by inhibiting the cytochromeP450-mediated catabolism of retinoic acids.

DNA methylation changes are among the most common abnormalities in humanneoplasia. Hypermethylation within the promotors of selected genes isusually associated with inactivation of the involved genes. The term“DNA methyl transferase inhibitors” is used to indicate compounds thatact through pharmacological inhibition of DNA methyl transferase andreactivation of tumour suppressor gene expression.

The term “kinase inhibitors” comprises potent inhibitors of kinases thatare involved in cell cycle progression and programmed cell death(apoptosis)

The term “farnesyltransferase inhibitors” is used to indicate compoundsthat were designed to prevent farnesylation of Ras and otherintracellular proteins. They have been shown to have effect on malignantcell proliferation and survival.

The term “other HDAC inhibitors” comprises but is not limited to:

-   -   short-chain fatty acids for example butyrate, 4-phenylbutyrate        or valproic acid;    -   hydroxamic acids for example suberoylanilide hydroxamic acid        (SAHA), biaryl hydroxamate A-161906, bicyclic        aryl-N-hydroxycarboxamides, pyroxamide, CG-1521, PXD-101,        sulfonamide hydroxamic acid, LAQ-824, trichostatin A (TSA),        oxamflatin, scriptaid, m-carboxy cinnamic acid bishydroxamic        acid, or trapoxin-hydroxamic acid analogue;    -   cyclic tetrapeptides for example trapoxin, apidicin or        depsipeptide;    -   benzamides for example MS-275 or CI-994, or    -   depudecin.

For the treatment of cancer the compounds according to the presentinvention may be administered to a patient as described above, inconjunction with irradiation. Irradiation means ionising radiation andin particular gamma radiation, especially that emitted by linearaccelerators or by radionuclides that are in common use today. Theirradiation of the tumour by radionuclides can be external or internal.

The present invention also relates to a combination according to theinvention of an anti-cancer agent and a HDAC inhibitor according to theinvention.

The present invention also relates to a combination according to theinvention for use in medical therapy for example for inhibiting thegrowth of tumour cells.

The present invention also relates to a combinations according to theinvention for inhibiting the growth of tumour cells.

The present invention also relates to a method of inhibiting the growthof tumour cells in a human subject which comprises administering to thesubject an effective amount of a combination according to the invention.

This invention further provides a method for inhibiting the abnormalgrowth of cells, including transformed cells, by administering aneffective amount of a combination according to the invention.

The other medicinal agent and HDAC inhibitor may be administeredsimultaneously (e.g. in separate or unitary compositions) orsequentially in either order. In the latter case, the two compounds willbe administered within a period and in an amount and manner that issufficient to ensure that an advantageous or synergistic effect isachieved. It will be appreciated that the preferred method and order ofadministration and the respective dosage amounts and regimes for eachcomponent of the combination will depend on the particular othermedicinal agent and HDAC inhibitor being administered, their route ofadministration, the particular tumour being treated and the particularhost being treated. The optimum method and order of administration andthe dosage amounts and regime can be readily determined by those skilledin the art using conventional methods and in view of the information setout herein.

The platinum coordination compound is advantageously administered in adosage of 1 to 500 mg per square meter (mg/m²) of body surface area, forexample 50 to 400 mg/m², particularly for cisplatin in a dosage of about75 mg/m² and for carboplatin in about 300 mg/m² per course of treatment.

The taxane compound is advantageously administered in a dosage of 50 to400 mg per square meter (mg/m²) of body surface area, for example 75 to250 mg/m², particularly for paclitaxel in a dosage of about 175 to 250mg/m² and for docetaxel in about 75 to 150 mg/m² per course oftreatment.

The camptothecin compound is advantageously administered in a dosage of0.1 to 400 mg per square meter (mg/m²) of body surface area, for example1 to 300 mg/m², particularly for irinotecan in a dosage of about 100 to350 mg/m² and for topotecan in about 1 to 2 mg/m² per course oftreatment.

The anti-tumor podophyllotoxin derivative is advantageously administeredin a dosage of 30 to 300 mg per square meter (mg/m²) of body surfacearea, for example 50 to 250 mg/m², particularly for etoposide in adosage of about 35 to 100 mg/m² and for teniposide in about 50 to 250mg/m² per course of treatment.

The anti-tumor vinca alkaloid is advantageously administered in a dosageof 2 to 30 mg per square meter (mg/m²) of body surface area,particularly for vinblastine in a dosage of about 3 to 12 mg/m², forvincristine in a dosage of about 1 to 2 mg/m², and for vinorelbine indosage of about 10 to 30 mg/m² per course of treatment.

The anti-tumor nucleoside derivative is advantageously administered in adosage of 200 to 2500 mg per square meter (mg/m²) of body surface area,for example 700 to 1500 mg/m², particularly for 5-FU in a dosage of 200to 500 mg/m², for gemcitabine in a dosage of about 800 to 1200 mg/m² andfor capecitabine in about 1000 to 2500 mg/m² per course of treatment.

The alkylating agents such as nitrogen mustard or nitrosourea isadvantageously administered in a dosage of 100 to 500 mg per squaremeter (mg/m²) of body surface area, for example 120 to 200 mg/m²,particularly for cyclophosphamide in a dosage of about 100 to 500 mg/m²,for chlorambucil in a dosage of about 0.1 to 0.2 mg/kg, for carmustinein a dosage of about 150 to 200 mg/m², and for lomustine in a dosage ofabout 100 to 150 mg/m² per course of treatment.

The anti-tumor anthracycline derivative is advantageously administeredin a dosage of 10 to 75 mg per square meter (mg/m²) of body surfacearea, for example 15 to 60 mg/m², particularly for doxorubicin in adosage of about 40 to 75 mg/m², for daunorubicin in a dosage of about 25to 45 mg/m², and for idarubicin in a dosage of about 10 to 15 mg/m² percourse of treatment.

Trastuzumab is advantageously administered in a dosage of 1 to 5 mg persquare meter (mg/m²) of body surface area, particularly 2 to 4 mg/m² percourse of treatment.

The antiestrogen agent is advantageously administered in a dosage ofabout 1 to 100 mg daily depending on the particular agent and thecondition being treated. Tamoxifen is advantageously administered orallyin a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day,continuing the therapy for sufficient time to achieve and maintain atherapeutic effect. Toremifene is advantageously administered orally ina dosage of about 60 mg once a day, continuing the therapy forsufficient time to achieve and maintain a therapeutic effect.Anastrozole is advantageously administered orally in a dosage of about 1mg once a day. Droloxifene is advantageously administered orally in adosage of about 20-100 mg once a day. Raloxifene is advantageouslyadministered orally in a dosage of about 60 mg once a day. Exemestane isadvantageously administered orally in a dosage of about 25 mg once aday.

These dosages may be administered for example once, twice or more percourse of treatment, which may be repeated for example every 7,14, 21 or28 days.

In view of their useful pharmacological properties, the components ofthe combinations according to the invention, i.e. the other medicinalagent and the HDAC inhibitor may be formulated into variouspharmaceutical forms for administration purposes. The components may beformulated separately in individual pharmaceutical compositions or in aunitary pharmaceutical composition containing both components.

The present invention therefore also relates to a pharmaceuticalcomposition comprising the other medicinal agent and the HDAC inhibitortogether with one or more pharmaceutical carriers.

The present invention also relates to a combination according to theinvention in the form of a pharmaceutical composition comprising ananti-cancer agent and a HDAC inhibitor according to the inventiontogether with one or more pharmaceutical carriers.

The present invention further relates to the use of a combinationaccording to the invention in the manufacture of a pharmaceuticalcomposition for inhibiting the growth of tumour cells.

The present invention further relates to a product containing as firstactive ingredient a HDAC inhibitor according to the invention and assecond active ingredient an anticancer agent, as a combined preparationfor simultaneous, separate or sequential use in the treatment ofpatients suffering from cancer.

Experimental Part

The following examples are provided for purposes of illustration. “BSA”means bovine serum albumine, “DCM” means dichloromethane, “DIEA” meansdiisopropylethylamine, “DMF” means dimethylformamide, “DMSO” meansdimethylsulfoxide, “EtOAc” means ethyl acetate, “Fmoc” meansfluorenylmethoxycarbonyl, “Hepes” means4-(-2-hydroxyethyl)-1-piperazine-ethanesulfonic acid, “HOBT” means1-hydroxy-1H-benzotriazole, “MeOH” means methanol, “PyBop” meansbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate,“PyBrOP” means bromo-tris-pyrrolidino-phosphonium hexafluorophosphate,“TEA” means triethylamine, “TFA” means trifluoroacetic acid “THF” meanstetrahydrofuran, Extrelut™ is a product of Merck KgaA, Darmstadt,Germany, and is a short column comprising diatomaceous earth.

A. Preparation of the Intermediates

EXAMPLE A1

a) Preparation of

A solution of 1-(phenylmethyl)-piperazine (0.068 mol) in acetonitrilep.a. (135 ml) was added gradually to a solution of potassium carbonate(0.18 mol) and 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethylester (0.082 mol) in acetonitrile p.a. (135 ml) and the reaction mixturewas stirred for 45 min at room temperature. Then, the reaction mixturewas stood overnight. DCM (400 ml) was added. Water (300 ml) was addedand the organic layer was separated, dried (MgSO₄), filtered and thesolvent was evaporated. The residue (28 g) was purified by columnchromatography over silica gel (eluent: DCM/MeOH 95/5). The purefractions were collected and the solvent was evaporated. The residue wascrystallized from acetonitrile, filtered off and dried in vacuo,yielding 15.1 g of intermediate 1.

b) Preparation of

A mixture of intermediate 1 (0.03 mol) in EtOH (250 ml) was hydrogenatedat 50° C. with Pd/C 10% (2 g) as a catalyst. After uptake of H₂ (1equiv), the catalyst was filtered off and the filtrate was evaporated.The residue was purified by column chromatography over silica gel(eluent: DCM/(MeOH/NH₃) 90/10). The product fractions were collected andthe solvent was evaporated, yielding 6.8 g (>96%) of intermediate 2.

c) Preparation of

A solution of dimethyl-sulfamoyl chloride (0.0015 mol) in DCM (1 ml) wasadded at 5° C. to a mixture of intermediate 2 (0.0012 mol) and TEA(0.0017 mol) in DCM (1 ml) under N₂ flow. The mixture was stirred atroom temperature for 18 hours. Potassium carbonate 10% was added. Themixture was extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated till dryness. Theresidue (0.69 g) was taken up in diethyl ether. The precipitate wasfiltered off and dried, yielding 0.64 g (73%) of intermediate 3, meltingpoint 193° C.

EXAMPLE A2

Preparation of

A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethylester (0.0434 mol) in acetonitrile (100 ml) was added dropwise at 10° cto a solution of 4-piperidinemethanamine (0.0868 mol) and potassiumcarbonate (0.0434 mol) in acetonitrile (200 ml) under N₂ flow. Themixture was stirred at room temperature for 2 hours, poured out into icewater and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (14.18 g)was purified by column chromatography over silica gel (20-45 μm)(eluent:DCM/MeOH/NH₄OH 90/10/1 to 80/20/2). The pure fractions were collectedand the solvent was evaporated, yielding 3.7 g (32%) of intermediate 4.

EXAMPLE A3

a) Preparation of

A mixture of intermediate 2 (0.0002 mol), (-phenyl-benzeneacetylchloride (0.0003 mol) and morpholinomethyl-PS-scavenger (SupplierNovabiochem cat No 01-64-0171: Morpholinomethyl polystyrene HL (200-400mesh), 2% divinylbenzene) (0.150 g) in DCM (5 ml) was stirred at roomtemperature for 20 hours, then tris(2-aminoethyl)amine-PS-scavenger(Supplier Novabiochem cat No 01-64-0170: Tris-(2-aminomethyl)-aminepolystyrene HL(200-400 mesh), 1% divinylbenzene) (0.150 g) was added andthe reaction mixture was stirred for another 4 hours. The scavengerswere filtered off, washed with DCM and the solvent was evaporated,yielding intermediate 5.

b) Preparation of

A mixture of intermediate 5 (0.0003 mol) in sodium hydroxide 1N (1.5ml), THF (4 ml) and MeOH (1 ml) was stirred at room temperature for 3days, then the reaction mixture was neutralised with HCl (1.5 ml, 1N).The mixture was filtered through Extrelut™ NT (supplier: Merck) anddried under N₂-flow, yielding intermediate 6.

c) Preparation of

A mixture of intermediate 6 (0.0003 mol),HOBT-6-carboxamidomethyl-PS-scavenger (0.200 g; Novabiochem Cat. No.01-64-0425) and N,N-dimethyl-4-pyridinamine (0.00015 mol) in DCM/DMF (5ml) was stirred at room temperature for 15 min., thenN,N′-methanetetraylbis-2-propanamine (0.070 ml) was added and thereaction mixture was shaken for 4 hours. The resin was washed 3 timeswith DCM, 3 times with DMF and again 3 times with DCM and 3 times withDMF, finally 6 times with DCM. A solution ofO-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.00026 mol) in DCM (5 ml)was added and the reaction mixture was shaken for 20 hours, then PSlinked methylisocyanate (Supplier Novabiochem cat No 01-64-0289:Methylisothiocyanate polystyrene HL(200-400 mesh), 2% divinylbenzene)(0.150 g) was added and the mixture was shaken for 4 hours. Thescavengers were filtered off, washed 2 times with DCM and the filtratewas used, yielding intermediate 7.

B. Preparation of the Final Compounds EXAMPLE B1

N-Fmoc-hydroxylamine 2-chlorotrityl resin (Novabiochem, 01-64-0165) wasdeprotected by 50% piperidine in DMF (RT, 24 hr). The resin was washedseveral times with DCM and DMF and swelled in DMF. Two equivalents ofacid¹, PyBrOP and 4 equivalents of DIEA were added as one portion. Themixture was shaken for 24 hr, liquid was drained and the resin waswashed several times by DCM and DMF. The resin was swelled in DMFcontaining 2 equivalents of amine, was shaken 24 hr at RT, the liquidwas drained and the resin was washed by DCM and DMF. An arylsulfonylchloride (2 eq.) was added as one portion to the resin swelled in DMFwith 4 equivalents of TEA. Reaction was stirred overnight, drained andthe resin was washed by DCM and DMF. The final product was cleaved by 5%TFA in DCM, analyzed by HPLC and MS and evaporated in the pre-weightedtest-tubes. ¹. Based on the loading of the resin.

For illustrative purposes the scheme hereunder is included.

EXAMPLE B2

N-Fmoc-hydroxylamine 2-chlorotrityl resin (Novabiochem, 01-64-0165) wasdeprotected by 50% piperidine in DMF (RT, 24 hr)¹. The resin was washed²several times with DCM and DMF and swelled in DMF. Two equivalents ofacid³, PyBrOP⁴ and 4 equivalents of DIEA were added as one portion. Themixture was shaken for 24 hr, liquid was drained and the resin waswashed several times by DCM and DMF. The resin was swelled in DMFcontaining 2 equivalents of amine, was shaken 24 hr at RT, the liquidwas drained and the resin was washed by DCM and DMF. The final productwas cleaved by 5% TFA in DCM, analyzed by HPLC and MS and evaporated inthe pre-weighted test-tubes. ¹. In one example compound 1carboxymethanethiol 4-methoxytrityl resin (Novabiochem, 01-64-0238) wasused.². In one case also MeOH was used in the different washingprocedures compound 1.³. Based on the loading of the resin.⁴. In onecase PyBrOP was replaced by PyBOP compound 1.

EXAMPLE B3

N-Fmoc-hydroxylamine 2-chlorotrityl resin (Novabiochem, 01-64-0165) wasdeprotected by 50% piperidine in DMF (RT, 24 hr)¹. The resin was washed²several times with DCM and DMF and swelled in DMF. Two equivalents ofacid³, PyBrOP⁴ and 4 equivalents of DIEA were added as one portion. Themixture was shaken for 24 hr, liquid was drained and the resin waswashed several times by DCM and DMF. The resin was swelled in DMFcontaining 2 equivalents of amine, was shaken 24 hr at RT, the liquidwas drained and the resin was washed by DCM and DMF. Three equivalentsof acid, DIC and DIEA were shaken with resin overnight at RT. The resinwas drained and washed by DCM and DMF. The final product was cleaved by5% TFA in DCM, analyzed by HPLC and MS and evaporated in thepre-weighted test-tubes.

EXAMPLE B4

a) Preparation of

A mixture of intermediate 3 (0.0016 mol) and sodium hydroxide (0.0033mol) in EtOH (6 ml) was stirred and refluxed for 2 hours, then cooled toroom temperature. The precipitate was filtered, washed with EtOH anddried, yielding 0.59 g (>100%) of intermediate 8 .Na.

b) Preparation of

N′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride (0.0021 mol) was added portionwise to a mixture ofintermediate 8 .Na (0.0016 mol),O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0021 mol) and1-hydroxy-1H-benzotriazole (0.0021 mol) in DCM/THF (10 ml) under N₂flow. The mixture was stirred at room temperature for a week end.Potassium carbonate 10% was added. The mixture was extracted with DCM.The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated till dryness. The residue (0.94 g) was purifiedby column chromatography over kromasil (eluent: DCM/MeOH/NH₄OH 97/3/0.1;15-40 μm). The pure fractions were collected and the solvent wasevaporated. The residue (0.45 g, 65%) was taken up in diethyl ether. Theprecipitate was filtered off and dried, yielding 0.422 g (61%) ofintermediate 9, melting point 183° C.

c) Preparation of

Trifluoroacetic acid (0.5 ml) was added to a mixture of intermediate 9(0.0009 mol) in MeOH (10 ml). The mixture was stirred at roomtemperature for 18 hours. The precipitate was filtered, washed with DCMand dried., yielding 0.176 g (59%) of compound 2, melting point >260° C.

EXAMPLE B5

Preparation of

A mixture of intermediate 2 (0.0019 mol) and sulfamide (0.0021 mol) in1,2-dimethoxy-ethane (5 ml) was stirred and refluxed for 4 days. Waterwas added. The mixture was filtered off and dried, yielding 0.51 g (83%)of intermediate 10, melting point 192° C.

Intermediate 10 was handled analogously as described in example [B4] togive 0.034 g (13%) of compound 3, melting point 212° C.

EXAMPLE B6

Preparation of

A solution of dimethyl-sulfamoyl chloride (0.007 mol) in DCM (5 ml) wasadded at 10° C. to a solution of intermediate 4 (0.0057 mol) and TEA(0.0085 mol) in DCM (5 ml) under N₂ flow. The mixture was stirredovernight, poured out into ice water and extracted with DCM. The organiclayer was separated, dried (MgSO₄), filtered, and the solvent wasevaporated. The residue was crystallized from CH₃CN/diethyl ether. Theprecipitate was filtered off and dried, yielding 0.492 g (24%) ofintermediate 11, melting point 142° C.

Intermediate 11 was handled analogously as described in example [B4] togive 0.7 g (85%) of compound 4, melting point 182° C.

EXAMPLE B7

Preparation of

A mixture of intermediate 7 (0.0003 mol) in acetic acid, trifluoro-acetic acid (5 ml, 5% in MeOH) was stirred at room temperature for 20hours, then the reaction mixture was blown dry, yielding compound 5.

EXAMPLE B8

Preparation of

A mixture of intermediate 2 (0.0025 mol), 2-naphthalenecarbonyl chloride(0.003 mol) and potassium carbonate (0.005 mol) in acetonitrile (20 ml)was stirred and refluxed overnight, then cooled to room temperature,poured out into ice water and extracted with DCM. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated. Theresidue was crystallized from diethyl ether. The precipitate wasfiltered off and dried, yielding 0.97 g (100%) of intermediate 12,melting point 140° C. Intermediate 12 was handled analogously asdescribed in example [B4] to give 0.338 g (86%) of compound 6, meltingpoint 130° C.

Table F-1 lists the compounds that were prepared according to one of theabove Examples. The following abbreviations were used in the tables.Co.No. stands for Compound Number, Ex. [Bn^(o)] referred to the samemethod as described in the Bn^(o) examples, C₂HF₃O₂ stands for thetrifluoroacetate salt. Some compounds have been characterized viamelting point (mp.), other compounds were characterized via MassSpectral data [MH⁺] (ms.).

TABLE F-1

C₂HF₃O₂ (1:1), Co. No. 1; Ex. [B2]

Co. No. 2; Ex. [B4]; mp >260° C.

Co. No. 3; Ex. [B5]; mp 212° C.

Co. No. 4; Ex. [B6]; mp 182° C.

•C₂HF₃O₂ (1:2), Co. No. 7; Ex. [B2]; ms. 412

•C₂HF₃O₂ (1:2), Co. No. 8; Ex. [B2]; ms. 383

•C₂HF₃O₂ (1:2), Co. No. 9; Ex. [B2]; ms. 383

•C₂HF₃O₂ (1:1), Co. No. 10; Ex. [B2]; ms. 361

•C₂HF₃O₂ (1:1), Co. No. 11; Ex. [B2]; ms. 314

•C₂HF₃O₂ (1:1), Co. No. 12; Ex. [B3]; ms. 366

•C₂HF₃O₂ (1:1), Co. No. 13; Ex. [B2]; ms. 314

•C₂HF₃O₂ (1:1), Co. No. 14; Ex. [B2]; ms. 412

C₂HF₃O₂ (1:2), Co. No. 15; Ex. [B2]; ms. 281

C₂HF₃O₂ (1:2), Co. No. 16; Ex. [B2]; ms. 365

C₂HF₃O₂ (1:2), Co. No. 17; Ex. [B2]; ms. 295

Co. No. 18; Ex. [B1]; ms. 398

Co. No. 19; Ex. [B1]; ms. 328

Co. No. 20; Ex. [B1]; ms. 300

Co. No. 21; Ex. [B1]; ms. 329

Co. No. 22; Ex. [B1]; ms. 354

Co. No. 5; Ex. [B7]; ms. 418

Co. No. 6; Ex. [B8]; mp. 130° C.

Co. No. 23; Ex. [B7]; ms. 348

Co. No. 24; Ex. [B7]; ms. 378

Co. No. 25; Ex. [B7]; ms. 329

Co. No. 26; Ex. [B7]; ms. 402

Co. No. 27; Ex. [B7]; ms. 329

C. PHARMACOLOGICAL EXAMPLE

The in vitro assay for inhibition of histone deacetylase (see exampleC.1) measures the inhibition of HDAC enzymatic activity obtained withthe compounds of formula (I).

Cellular activity of the compounds of formula (I) was determined onA2780 tumour cells using a calorimetric assay for cell toxicity orsurvival (Mosmann Tim, Journal of Immunological Methods 65: 55-63,1983)(see example C.2).

Kinetic solubility in aqueous media measures the ability of a compoundto stay in aqueous solution upon dilution (see example C.3).

DMSO-stock solutions are diluted with a single aqueous buffer solvent in3 consecutive steps. For every dilution turbidity is measured with anephelometer.

A drug's permeability expresses its ability to move from one medium intoor through another. Specifically its ability to move through theintestinal membrane into the blood stream and/or from the blood streaminto the target. Permeability (see example C.4) can be measured throughthe formation of a filter-immobilized artificial membrane phospholipidbilayer. In the filter-immobilized artificial membrane assay, a“sandwich” is formed with a 96-well microtitre plate and a 96-wellfilter plate, such that each composite well is divided into two chamberswith a donor solution at the bottom and an acceptor solution at the top,separated by a 125 μm micro-filter disc (0.45 μm pores), coated with 2%(wt/v) dodecane solution of dioleoylphosphatidyl-choline, underconditions that multi-lamellar bilayers form inside the filter channelswhen the system contacts an aqueous buffer solution. The permeability ofcompounds through this artificial membrane is measured in cm/s. Thepurpose is to look for the permeation of the drugs through a parallelartificial membrane at 2 different pH's: 4.0 and 7.4. Compound detectionis done with UV-spectrometry at optimal wavelength between 250 and 500nm.

Metabolism of drugs means that a lipid-soluble xenobiotic or endobioticcompound is enzymatically transformed into (a) polar, water-soluble, andexcretable metabolite(s). The major organ for drug metabolism is theliver. The metabolic products are often less active than the parent drugor inactive. However, some metabolites may have enhanced activity ortoxic effects. Thus drug metabolism may include both “detoxication” and“toxication” processes. One of the major enzyme systems that determinethe organism's capability of dealing with drugs and chemicals isrepresented by the cytochrome P450 monooxygenases, which are NADPHdependent enzymes. Metabolic stability of compounds can be determined invitro with the use of subcellular human tissue (see example C.5). Heremetabolic stability of the compounds is expressed as % of drugmetabolised after 15 minutes incubation of these compounds withmicrosomes. Quantitation of the compounds was determined by LC-MSanalysis.

The tumour suppressor p53 transcriptionally activates a number of genesincluding the WAF1/CIP1 gene in response to DNA damage. The 21 kDaproduct of the WAF1 gene is found in a complex involving cyclins, cyclindependent kinases (CDKs), and proliferating cell nuclear antigen (PCNA)in normal cells but not transformed cells and appears to be a universalinhibitor of CDK activity. One consequence of p21WAF1 binding to andinhibiting CDKs is to prevent CDK-dependent phosphorylation andsubsequent inactivation of the Rb protein, which is essential for cellcycle progression. Induction of p21WAF1 in response to cellular contactwith a HDAC inhibitor is therefore a potent and specific indicator ofinhibition of cell cycle progression at both the G1 and G2 checkpoints.

The capacity of the compounds to induce p21WAF1 was measured with thep21WAF1 enzyme linked immunosorbent assay (WAF1 ELISA of Oncogene). Thep21WAF1 assay is a “sandwich” enzyme immunoassay employing both mousemonoclonal and rabbit polyclonal antibodies. A rabbit polyclonalantibody, specific for the human WAF1 protein, has been immobilized ontothe surface of the plastic wells provided in the kit. Any p21WAF presentin the sample to be assayed will bind to the capture antibody. Thebiotinylated detector monoclonal antibody also recognizes human p21WAF1protein, and will bind to any p21WAF1, which has been retained by thecapture antibody. The detector antibody, in turn, is bond by horseradishperoxidas-conjugated streptavidin. The horseradish peroxidase catalysesthe conversion of the chromogenic substrate tetra-methylbenzidine from acolorless solution to a blue solution (or yellow after the addition ofstopping reagent), the intensity of which is proportional to the amountof p21WAF1 protein bond to the plate. The colored reaction product isquantified using a spectrophotometer. Quantitation is achieved by theconstruction of a standard curve using known concentrations of p21WAF1(provided lyophilised)(see example C.6).

EXAMPLE C.1 In Vitro Assay for Inhibition of histone deacetylase

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg/ml with2×10⁻⁸ M of radiolabeled peptide substrate. As a substrate for measuringHDAC activity a synthetic peptide, i.e. the amino acids 14-21 of histoneH4, was used. The substrate is biotinylated at the NH₂-terminal partwith a 6-aminohexanoic acid spacer, and is protected at theCOOH-terminal part by an amide group and specifically [³H]acetylated atlysine 16. The substrate,biotin-(6-aminohexanoic)Gly-Ala-([³H]-acetyl-Lys-Arg-His-Arg-Lys-Val-NH₂),was added in a buffer containing 25 mM Hepes, 1 M sucrose, 0.1 mg/ml BSAand 0.01% Triton X-100 at pH 7.4. After 30 min the deacetylationreaction was terminated by the addition of HCl and acetic acid. (finalconcentration 0.035 mM and 3.8 mM respectively). After stopping thereaction, the free ³H-acetate was extracted with ethylacetate. Aftermixing and centrifugation, the radioactivity in an aliquot of the upper(organic) phase was counted in a β-counter.

For each experiment, controls (containing HeLa nuclear extract and DMSOwithout compound), a blank incubation (containing DMSO but no HeLanuclear extract or compound) and samples (containing compound dissolvedin DMSO and HeLa nuclear extract) were run in parallel. In firstinstance, compounds were tested at a concentration of 10⁻⁵M. When thecompounds showed activity at 10⁻⁵M, a concentration-response curve wasmade wherein the compounds were tested at concentrations between 10⁻⁵Mand 10⁻¹²M. In each test the blank value was substracted from both thecontrol and the sample values. The control sample represented 100% ofsubstrate deactylation. For each sample the radioactivity was expressedas a percentage of the mean value of the controls. When appropriateIC₅₀-values (concentration of the drug, needed to reduce the amount ofmetabolites to 50% of the control) were computed using probit analysisfor graded data. Herein the effects of test compounds are expressed aspIC₅₀ (the negative log value of the IC₅₀-value). All tested compoundsshowed enzymatic activity at a test concentration of 10⁻⁵M and 21compounds had a pIC₅₀≧5 (see table F-2).

EXAMPLE C.2 Determination of Antiproliferative Activity on A2780 Cells

All compounds tested were dissolved in DMSO and further dilutions weremade in culture medium. Final DMSO concentrations never exceeded 0.1%(v/v) in cell proliferation assays. Controls contained A2780 cells andDMSO without compound and blanks contained DMSO but no cells. MTT wasdissolved at 5 mg/ml in PBS. A glycine buffer comprised of 0.1 M glycineand 0.1 M NaCl buffered to pH 10.5 with NaOH (1 N) was prepared (allreagents were from Merck).

The human A2780 ovarian carcinoma cells (a kind gift from Dr. T. C.Hamilton [Fox Chase Cancer Centre, Pennsylvania, USA]) were cultured inRPMI 1640 medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamicinand 10% fetal calf serum. Cells were routinely kept as monolayercultures at 37° C. in a humidified 5% CO₂ atmosphere. Cells werepassaged once a week using a trypsin/EDTA solution at a split ratio of1:40. All media and supplements were obtained from Life Technologies.Cells were free of mycoplasma contamination as determined using theGen-Probe Mycoplasma Tissue Culture kit (supplier: BioMérieux).

Cells were seeded in NUNC™ 96-well culture plates (Supplier: LifeTechnologies) and allowed to adhere to the plastic overnight. Densitiesused for plating were 1500 cells per well in a total volume of 200 μlmedium. After cell adhesion to the plates, medium was changed and drugsand/or solvents were added to a final volume of 200 μl. Following fourdays of incubation, medium was replaced by 200 μl fresh medium and celldensity and viability was assessed using an MTT-based assay. To eachwell, 25 μl MTT solution was added and the cells were further incubatedfor 2 hours at 37° C. The medium was then carefully aspirated and theblue MTT-formazan product was solubilized by addition of 25 μl glycinebuffer followed by 100 μl of DMSO. The microtest plates were shaken for10 min on a microplate shaker and the absorbance at 540 nm was measuredusing an Emax 96-well spectrophotometer (Supplier: Sopachem).

Within an experiment, the results for each experimental condition arethe mean of 3 replicate wells. For initial screening purposes, compoundswere tested at a single fixed concentration of 10⁻⁶ M. For activecompounds, the experiments were repeated to establish fullconcentration-response curves. For each experiment, controls (containingno drug) and a blank incubation (containing no cells or drugs) were runin parallel. The blank value was subtracted from all control and samplevalues. For each sample, the mean value for cell growth (in absorbanceunits) was expressed as a percentage of the mean value for cell growthof the control. When appropriate, IC₅₀-values (concentration of thedrug, needed to reduce cell growth to 50% of the control) were computedusing probit analysis for graded data (Finney, D. J., Probit Analyses,2^(nd) Ed. Chapter 10, Graded Responses, Cambridge University Press,Cambridge 1962). Herein the effects of test compounds are expressed aspIC₅₀ (the negative log value of the IC₅₀-value). Most of the testedcompounds showed cellular activity at a test concentration of 10⁻⁶ M and9 compounds had a pIC₅₀≧5 (see table F-2)

EXAMPLE C.3 Kinetic Solubility in Aqueous Media

In the first dilution step, 10 μl of a concentrated stock-solution ofthe active compound, solubilized in DMSO (5 mM), was added to 100 μlphosphate citrate buffer pH 7.4 and mixed. In the second dilution step,an aliquot (20 μl) of the first dilution step was further dispensed in100 μl phosphate citrate buffer pH 7.4 and mixed. Finally, in the thirddilution step, a sample (20 μl) of the second dilution step was furtherdiluted in 100 μl phosphate citrate buffer pH 7.4 and mixed. Alldilutions were performed in 96-well plates. Immediately after the lastdilution step the turbidity of the three consecutive dilution steps weremeasured with a nephelometer. Dilution was done in triplicate for eachcompound to exclude occasional errors. Based on the turbiditymeasurements a ranking is performed into 3 classes. Compounds with highsolubility obtained a score of 3 and for this compounds the firstdilution is clear. Compounds with medium solubility obtained a score of2. For these compounds the first dilution is unclear and the seconddilution is clear. Compounds with low solubility obtained a score of 1and for these compounds both the first and the second dilution areunclear. The solubility of 9 compounds was measured. From thesecompounds 7 showed a score of 3, and 2 demonstrated a score of 1 (seetable F-2).

EXAMPLE C.4 Parallel Artificial Membrane Permeability Analysis

The stock samples (aliquots of 10 μl of a stock solution of 5 mM in 100%DMSO) were diluted in a deep-well or Pre-mix plate containing 2 ml of anaqueous buffer system pH 4 or pH 7.4 (PSR4 System Solution Concentrate(pION)).

Before samples were added to the reference plate, 150 μl of buffer wasadded to wells and a blank UV-measurement was performed. Thereafter thebuffer was discarded and the plate was used as reference plate. Allmeasurements were done in UV-resistant plates (supplier: Costar orGreiner).

After the blank measurement of the reference plate, 150 μl of thediluted samples was added to the reference plate and 200 μl of thediluted samples was added to donorplate 1. An acceptor filter plate 1(supplier: Millipore, type:MAIP N45) was coated with 4 μl of theartificial membrane-forming solution(1,2-Dioleoyl-sn-Glycer-3-Phosphocholine in Dodecane containing 0.1%2,6-Di-tert-butyl-4-methylphenol and placed on top of donor plate 1 toform a “sandwich”. Buffer (200 μl) was dispensed into the acceptor wellson the top. The sandwich was covered with a lid and stored for 18 h atroom temperature in the dark.

A blank measurement of acceptor plate 2 was performed through theaddition of 150 μl of buffer to the wells, followed by anUV-measurement. After the blank measurement of acceptor plate 2 thebuffer was discarded and 150 μl of acceptor solution was transferredfrom the acceptor filter plate 1 to the acceptor plate 2. Then theacceptor filter plate 1 was removed form the sandwich. After the blankmeasurement of donor plate 2 (see above), 150 μl of the donor solutionwas transferred from donor plate 1 to donor plate 2. The UV spectra ofthe donor plate 2, acceptor plate 2 and reference plate wells werescanned (with a SpectraMAX 190). All the spectra were processed tocalculate permeability with the PSR4p Command Software. All compoundswere measured in triplo. Carbamazepine, griseofulvin, acycloguanisine,atenolol, furosemide, and chlorothiazide were used as standards in eachexperiment. Compounds were ranked in 3 categories as having a lowpermeability (mean effect<0.5×10⁻⁶ cm/s; score 1), a medium permeability(1×10⁻⁶ cm/s>mean effect≧0.5×10⁻⁶ cm/s; score 2) or a high permeability(≧0.5×10⁻⁶ cm/s; score 3). Two compounds showed a score of 1 at one ofthe pH's measured.

EXAMPLE C.5 Metabolic Stability

Sub-cellular tissue preparations were made according to Gorrod et al.(Xenobiotica 5: 453-462, 1975) by centrifugal separation aftermechanical homogenization of tissue. Liver tissue was rinsed in ice-cold0.1 M Tris-HCl (pH 7.4) buffer to wash excess blood. Tissue was thenblotted dry, weighed and chopped coarsely using surgical scissors. Thetissue pieces were homogenized in 3 volumes of ice-cold 0.1 M phosphatebuffer (pH 7.4) using either a Potter-S (Braun, Italy) equipped with aTeflon pestle or a Sorvall Omni-Mix homogeniser, for 7×10 sec. In bothcases, the vessel was kept in/on ice during the homogenization process.

Tissue homogenates were centrifuged at 9000×g for 20 minutes at 4° C.using a Sorvall centrifuge or Beckman Ultracentrifuge. The resultingsupernatant was stored at −80° C. and is designated ‘S9’.

The S9 fraction can be further centrifuged at 100,000×g for 60 minutes(4° C.) using a Beckman ultracentrifuge. The resulting supernatant wascarefully aspirated, aliquoted and designated ‘cytosol’. The pellet wasre-suspended in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1ml per 0.5 g original tissue weight and designated ‘microsomes’.

All sub-cellular fractions were aliquoted, immediately frozen in liquidnitrogen and stored at −80° C. until use.

For the samples to be tested, the incubation mixture contained PBS(0.1M), compound (5 μM), microsomes (1 mg/ml) and a NADPH-generatingsystem (0.8 mM glucose-6-phosphate, 0.8 mM magnesium chloride and 0.8Units of glucose-6-phosphate dehydrogenase). Control samples containedthe same material but the microsomes were replaced by heat inactivated(10 min at 95 degrees Celsius) microsomes. Recovery of the compounds inthe control samples was always 100%.

The mixtures were preincubated for 5 min at 37 degrees Celsius. Thereaction was started at timepoint zero (t=0) by addition of 0.8 mM NADPand the samples were incubated for 15 min (t=15). The reaction wasterminated by the addition of 2 volumes of DMSO. Then the samples werecentrifuged for 10 min at 900×g and the supernatants were stored at roomtemperature for no longer as 24 h before analysis. All incubations wereperformed in duplo. Analysis of the supernatants was performed withLC-MS analysis. Elution of the samples was performed on a Xterra MS C18(50×4.6 mm, 5 μm, Waters, US). An Alliance 2790 (Supplier: Waters, US)HPLC system was used. Elution was with buffer A (25 mM ammoniumacetate(pH 5.2) in H₂O/acetonitrile (95/5)), solvent B being acetonitrile andsolvent C methanol at a flow rate of 2.4 ml/min. The gradient employedwas increasing the organic phase concentration from 0% over 50% B and50% C in 5 min up to 100% B in 1 min in a linear fashion and organicphase concentration was kept stationary for an additional 1.5 min. Totalinjection volume of the samples was 25 μl.

A Quattro (supplier: Micromass, Manchester, UK) triple quadrupole massspectrometer fitted with and ESI source was used as detector. The sourceand the desolvation temperature were set at 120 and 350° C. respectivelyand nitrogen was used as nebuliser and drying gas. Data were acquired inpositive scan mode (single ion reaction). Cone voltage was set at 10 Vand the dwell time was 1 sec.

Metabolic stability was expressed as % metabolism of the compound after15 min of incubation in the presence of active microsomes

$\left( {E({act})} \right)\left( {{\% \mspace{14mu} {metabolism}} = {\quad{{100\; \%} - {\left( {\left( \frac{\begin{matrix}{{Total}\mspace{14mu} {Ion}\mspace{14mu} {Current}\mspace{14mu} \left( {T\; I\; C} \right)\mspace{14mu} {of}} \\{{{E({act})}\mspace{14mu} {at}\mspace{14mu} t} = 15}\end{matrix}\mspace{14mu}}{{T\; I\; C\mspace{14mu} {of}\mspace{14mu} {E({act})}\mspace{14mu} {at}\mspace{14mu} t} = 0} \right) \times 100} \right).}}}} \right.$

Compounds that had a percentage metabolism less than 20% were defined ashighly metabolic stable. Compound that had a metabolism between 20 and70% were defined as intermediately stable and compounds that showed apercentage metabolism higher than 70 were defined as low metabolicstable. Three reference compounds were always included whenever ametabolic stability screening was performed. Verapamil was included as acompound with low metabolic stability (% metabolism=73%). Cisapride wasincluded as a compound with medium metabolic stability (% metabolism45%) and propanol was included as a compound with intermediate to highmetabolic stability (25% metabolism). These reference compounds wereused to validate the metabolic stability assay.

One compound was tested and showed a percentage metabolism less than20%.

EXAMPLE C.6 p21 Induction Capacity

The following protocol has been applied to determine the p21 proteinexpression level in human A2780 ovarian carcinoma cells. The A2780 cells(20000 cells/180 μl) were seeded in 96 microwell plates in RPMI 1640medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamicin and 10%fetal calf serum. 24 hours before the lysis of the cells, compounds wereadded at final concentrations of 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ M. Allcompounds tested were dissolved in DMSO and further dilutions were madein culture medium. 24 hours after the addition of the compound, thesupernatants were removed from the cells. Cells were washed with 200 μlice-cold PBS. The wells were aspirated and 30 μl of lysisbuffer (50 mMTris.HCl (pH 7.6), 150 mM NaCl, 1% Nonidet p40 and 10% glycerol) wasadded. The plates were incubated overnight at −70° C.

The appropriate number of microtiter wells were removed from the foilpouch and placed into an empty well holder. A working solution (1×) ofthe Wash Buffer (20× plate wash concentrate: 100 ml 20-fold concentratedsolution of PBS and surfactant. Contains 2% chloroacetamide) wasprepared. The lyophilised p21WAF standard was reconstituted withdistilled H₂O and further diluted with sample diluent (provided in thekit)

The samples were prepared by diluting them 1:4 in sample diluent. Thesamples (100 μl) and the p21WAF1 standards (100 μl) were pipetted intothe appropriate wells and incubated at room temperature for 2 hours. Thewells were washed 3 times with 1× wash buffer and then 100 μl ofdetector antibody reagent (a solution of biotinylated monoclonal p21WAF1antibody) was pipetted into each well. The wells were incubated at roomtemperature for 1 hour and then washed three times with 1× wash buffer.The 400× conjugate (peroxidase streptavidine conjugate: 400-foldconcentrated solution) was diluted and 100 μl of the 1× solution wasadded to the wells. The wells were incubated at room temperature for 30min and then washed 3 times with 1× wash buffer and 1 time withdistilled H₂O. Substrate solution (chromogenic substrate)(100 μl) wasadded to the wells and the wells were incubated for 30 minutes in thedark at room temperature. Stop solution was added to each well in thesame order as the previously added substrate solution. The absorbance ineach well was measured using a spectrophotometric plate reader at dualwavelengths of 450/595 nm.

For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was substracted from all control and sample values. For eachsample, the value for p21WAF1 induction (in absorbance units) wasexpressed as the percentage of the value for p21WAF1 present in thecontrol. Percentage induction higher than 130% was defined assignificant induction. Three compounds were tested in this assay. Twoshowed significant induction.

TABLE F-2 Table F-2 lists the results of the compounds that were testedaccording to example C. 1, C. 2, and C. 3. Enzyme Cellular activityactivity Solubility Co. No. pIC50 pIC50 Score 8 >5 7 >5 9 >5 10 >5 11 >512 >5 1 <5 <5 1 18 6.173 6.166 1 5 7.096 6.181 23 6.932 5.796 24 7.0736.084 25 6.29 <5 26 6.984 5.378 27 6.433 <5 6 7.104 5.828 3 19 5.536 <53 20 5.451 <5 21 5.679 <5 3 22 5.599 5.297 3 2 6.615 5.534 3 3 6.881 <53 4 7.27 5.528 3

D. COMPOSITION EXAMPLE Film-Coated Tablets

Preparation of Tablet Core

A mixture of 100 g of a compound of formula (I), 570 g lactose and 200 gstarch is mixed well and thereafter humidified with a solution of 5 gsodium dodecyl sulphate and 10 g polyvinyl-pyrrolidone in about 200 mlof water. The wet powder mixture is sieved, dried and sieved again. Thenthere is added 100 g microcrystalline cellulose and 15 g hydrogenatedvegetable oil. The whole is mixed well and compressed into tablets,giving 10,000 tablets, each comprising 10 mg of a compound of formula(I).

Coating

To a solution of 10 g methyl cellulose in 75 ml of denaturated ethanolthere is added a solution of 5 g of ethyl cellulose in 150 ml ofdichloromethane. Then there are added 75 ml of dichloromethane and 2.5ml 1,2,3-propanetriol 10 g of polyethylene glycol is molten anddissolved in 75 ml of dichloromethane. The latter solution is added tothe former and then there are added 2.5 g of magnesium octadecanoate, 5g of polyvinyl-pyrrolidone and 30 ml of concentrated colour suspensionand the whole is homogenated. The tablet cores are coated with the thusobtained mixture in a coating apparatus.

1-12. (canceled)
 13. A compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein n is 0, 1, 2 or 3and when n is 0 then a direct bond is intended; each Q is

each X is

each Y is

each Z is nitrogen or

R¹ is —C(O)NR⁵R⁶, —N(H)C(O)R⁷, —C(O)—C₁₋₆alkanediylSR⁷, —NR⁸C(O)N(OH)R⁷,—NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R⁷ or anotherZn-chelating-group wherein R⁵ and R⁶ are each independently selectedfrom hydrogen, hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, aminoC₁₋₆alkyl oraminoaryl; R⁷ is independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆alkylcarbonyl, arylC₁₋₆alkyl, C₁₋₆alkylpyrazinyl, pyridinone,pyrrolidinone or methylimidazolyl; R⁸ is independently selected fromhydrogen or C₁₋₆alkyl; R² is hydrogen, halo, hydroxy, amino, nitro,C₁₋₆alkyl, C₁₋₆alkyloxy, trifluoromethyl, di(C₁₋₆alkyl)amino,hydroxyamino or naphtalenylsulfonylpyrazinyl; R³ is hydrogen, C₁₋₆alkyl,arylC₂₋₆alkenediyl, furanylcarbonyl, naphthalenylcarbonyl,—C(O)phenylR⁹, C₁₋₆alkylaminocarbonyl, aminosulfonyl, arylaminosulfonyl,aminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino,arylaminosulfonylamino, aminosulfonylaminoC₁₋₆alkyl,di(C₁₋₆alkyl)aminosulfonylaminoC₁₋₆alkyl,arylaminosulfonylaminoC₁₋₆alkyl, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,C₁₋₁₂alkylsulfonyl, di(C₁₋₆alkyl)aminosulfonyl,trihaloC₁₋₆alkylsulfonyl, di(aryl)C₁₋₆alkylcarbonyl,thiophenylC₁₋₆alkylcarbonyl, pyridinylcarbonyl or arylC₁₋₆alkylcarbonylwherein each R⁹ is independently selected from phenyl; phenylsubstituted with one, two or three substituents independently selectedfrom halo, amino, C₁₋₆alkyl, C₁₋₆alkyloxy, hydroxyC₁₋₄alkyl,hydroxyC₁₋₄alkyloxy, aminoC₁₋₄alkyloxy, di(C₁₋₄alkyl)aminoC₁₋₄alkyloxy,di(C₁₋₆alkyl)aminoC₁₋₆alkyl,di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl,hydroxyC₁₋₄alkylpiperazinylC₁₋₄alkyl, C₁₋₄alkyloxypiperidinylC₁₋₄alkyl,hydroxyC₁₋₄alkyloxyC₁₋₄alkylpiperazinyl, C₁₋₄alkylpiperazinylC₁₋₄alkyl,di(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkyl, pyrrolidinylC₁₋₄alkyloxy,morpholinylC₁₋₄alkyloxy, or morpholinylC₁₋₄alkyl; thiophenyl; orthiophenyl substituted with di(C₁₋₄alkyl)aminoC₁₋₄alkyloxy,di(C₁₋₆alkyl)aminoC₁₋₆alkyl,di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl,pyrrolidinylC₁₋₄alkyloxy, C₁₋₄alkylpiperazinylC₁₋₄alkyl,di(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkyl or morpholinylC₁₋₄alkyloxy; R⁴ ishydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkyloxy,arylC₁₋₆alkyl, aminocarbonyl, hydroxycarbonyl, aminoC₁₋₆alkyl,aminocarbonylC₁₋₆alkyl, hydroxycarbonylC₁₋₆alkyl, hydroxyaminocarbonyl,C₁₋₆alkyloxycarbonyl, C₁₋₆alkylaminoC₁₋₆alkyl ordi(C₁₋₆alkyl)aminoC₁₋₆alkyl; when R³ and R⁴ are present on the samecarbon atom, R³ and R⁴together may form a bivalent radical of formula—C(O)—NH—CH₂—NR¹⁰—  (a-1) wherein R¹⁰ is hydrogen or aryl; when R³ andR⁴ are present on adjacent carbon atoms, R³ and R⁴ together may form abivalent radical of formula═CH—CH═CH—CH═  (b-1); aryl in the above is phenyl, or phenyl substitutedwith one or more substituents each independently selected from halo,C₁₋₆alkyl, C₁₋₆alkyloxy, trifluoromethyl, cyano or hydroxycarbonyl. 14.A compound as claimed in claim 13 wherein R¹ is —C(O)NR⁵R⁶,—C(O)—C₁₋₆alkanediylSR⁷, —NR⁸C(O)N(OH)R⁷, —NR⁸C(O)C₁₋₆alkanediylSR⁷,—NR⁸C(O)C═N(OH)R⁷ or another Zn-chelating-group wherein R⁵ and R⁶ areeach independently selected from hydrogen, hydroxy, hydroxyC₁₋₆alkyl oraminoC₁₋₆alkyl; R² is hydrogen, halo, hydroxy, amino, nitro, C₁₋₆alkyl,C₁₋₆alkyloxy, trifluoromethyl or di(C₁₋₆alkyl)amino; R³ is hydrogen,C₁₋₆alkyl, arylC₂₋₆alkenediyl, furanylcarbonyl, naphthalenylcarbonyl,—C(O)phenylR⁹, C₁₋₆alkylaminocarbonyl, aminosulfonyl, arylaminosulfonyl,aminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino,di(C₁₋₆alkyl)aminoC₁₋₆alkyl, C₁₋₁₂alkylsulfonyl,di(C₁₋₆alkyl)aminosulfonyl or pyridinylcarbonyl wherein each R⁹ isindependently selected from phenyl; phenyl substituted with one, two orthree substituents independently selected from halo, C₁₋₆alkyl,C₁₋₆alkyloxy; or thiophenyl; and R⁴ is hydrogen, hydroxy, amino,hydroxyC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl,aminoC₁₋₆alkyl, C₁₋₆alkylaminoC₁₋₆alkyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl.15. A compound as claimed in claim 13 wherein n is 0 or 1; each R¹ is—C(O)NH(OH) or —NHC(O)C₁₋₆alkanediylSH; R² is hydrogen or nitro; R³ isC₁₋₆alkyl, arylC₂₋₆alkenediyl, furanylcarbonyl, naphthalenylcarbonyl,C₁₋₆alkylaminocarbonyl, aminosulfonyl,di(C₁₋₆alkyl)aminosulfonylaminoC₁₋₆alkyl, di(C₁₋₆alkyl)aminoC₁₋₆alkyl,C₁₋₁₂alkylsulfonyl, di(C₁₋₆alkyl)aminosulfonyl,trihaloC₁₋₆alkylsulfonyl, di(aryl)C₁₋₆alkylcarbonyl,thiophenylC₁₋₆alkylcarbonyl, pyridinylcarbonyl or arylC₁₋₆alkylcarbonyl;R⁴ is hydrogen; when R³ and R⁴ are present on the same carbon atom R³and R⁴ together may form a bivalent radical of formula (a-1) wherein R¹⁰is aryl; or when R³ and R⁴ are present on adjacent carbon atoms R³ andR⁴ together may form a bivalent radical of formula (b-1).
 16. A compoundas claimed in claim 13 wherein n is 1; each Z is nitrogen; R¹ is—C(O)NH(OH); R² is hydrogen; R³ is naphthalenylcarbonyl,C₁₋₁₂alkylsulfonyl or di(aryl)C₁₋₆alkylcarbonyl; and R⁴ is hydrogen. 17.A compound according to claim 13 selected from the following compounds


18. A pharmaceutical composition comprising pharmaceutically acceptablecarriers and as an active ingredient a therapeutically effective amountof a compound as claimed in claim
 13. 19. A process of preparing apharmaceutical composition as claimed in claim 18 wherein thepharmaceutically acceptable carriers and the compound are intimatelymixed.
 20. The method of treating proliferative disease comprisingadministering to a patient in need of such treatment, ananti-proliferative disease-effective amount of a compound of claim 13.21. A process for preparing a compound as claimed in claim 13,characterized by reacting an intermediate of formula (II) with anappropriate acid, such as for example, trifluoro acetic acid, yielding ahydroxamic acid of formula (I-a), wherein R¹ is —C(O)NH(OH)


22. A method of detecting or identifying a histone deactylase (HDAC) ina biological sample comprising detecting or measuring the formation of acomplex between a labelled compound as defined in claim 13 and a HDAC.23. A combination of anti-cancer agents and a compound of claim 13.